1. Field of the Invention
Hybrid DNA technology has revolutionized the ability to produce polypeptides of an infinite variety of compositions. Since living forms are composed of proteins and employ proteins for regulation, the ability to duplicate these proteins at will offers unique opportunities for investigating the manner in which these proteins function and the use of such proteins, fragments of such proteins, or analogs in therapy and diagnosis.
There have been numerous advances in improving the rate and amount of protein produced by a cell. Most of these advances have been associated with higher copy numbers, more efficient promoters, and means for reducing the amount of degradation of the desired product. It is evident that it would be extremely desirable to be able to secrete polypeptides of interest, where such polypeptides are the product of interest.
Furthermore, in many situations, the polypeptide of interest does not have an initial methionine amino acid. This is usually a result of there being a processing signal in the gene encoding for the polypeptide of interest, which the gene source recognizes and cleaves with an appropriate peptidase. Since in most situations, genes of interest are heterologous to the host in which the gene is to be expressed, such processing occurs imprecisely and in low yield in the expression host. In this case, while the protein which is obtained will be identical to the peptide of interest for almost all of its sequence, it will differ at the N-terminus which can deleteriously affect physiological activity.
There are, therefore, many reasons why it would be extremely advantageous to prepare DNA sequences, which would encode for the secretion and maturing of the polypeptide product Furthermore, where sequences can be found for processing, which result in the removal of amino acids superfluous to the polypeptide of interest, the opportunity exists for having a plurality of DNA sequences, either the same or different, in tandem, which may be encoded on a single transcript.
2. Description of the Prior Art
U.S. Pat. No. 4,336,336 describes for prokaryotes the use of a leader sequence coding for a noncytoplasmic protein normally transported to or beyond the cell surface, resulting in transfer of the fused protein to the periplasmic space. U.S. Pat. No. 4,338,397 describes for prokaryotes using a leader sequence which provides for secretion with cleavage of the leader sequence from the polypeptide sequence of interest. U.S. Pat. No. 4,338,397, columns 3 and 4, provide for useful definitions, which definitions are incorporated herein by reference.
Kurjan and Herskowitz, Cell (1982) 30:933-943 describes a putative .alpha.-factor precusor containing four tandem copies of mature .alpha.-factor, describing the sequence and postulating a processing mechanism. Kurjan and Herskowitz, Abstracts of Papers presented at the 1981 Cold Spring Harbor meeting on The Molecular Biology of Yeasts, page 242, in an Abstract entitled, "A Putative .alpha.-Factor Precursor Containing Four Tandem Repeats of Mature .alpha.-Factor," describe the sequence encoding for the .alpha.-factor and spacers between two of such sequences. Blair et al., Abstracts of Papers, ibid, page 243, in an Abstract entitled "Synthesis and Processing of Yeast Pheremones: Identification and Characterization of Mutants That Produce Altered .alpha.-Factors," describe the effect of various mutants on the production of mature o-factor.